There are several applications where it is desirable to quickly and accurately detect the presence of one or more known sequences in a target nucleic acid. Typically this is done using hybridization arrays, PCR, or short-range Sanger sequencing. All of these methods, however, require that one specify which sequences are to be detected (hybridization array), or know a priori the primer sequences in the target (PCR, Sanger).
Sanger sequencing reactions and related methods are usually analyzed by electrophoresis or mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has two primary advantages over electrophoretic methods for sequencing nucleic acids: high speed and high resolution (Nordhoff et al. 2000; Koster et al. 1996). The main disadvantage of MALDI-TOF mass spectrometry in this regard is its highly limited read length (15 to 40 bases), as compared to electrophoresis, which routinely gives read lengths of several hundred bases.
Recently developed mass spectrometric methods for diagnostic resequencing of DNA utilize a controlled fragmentation of the target DNA sequence, usually several hundred bases in length, into many smaller non-overlapping oligonucleotides of less than fifteen bases (Elso et al. 2002; Rodi et al. 2002). The mass spectrum of these fragments can be thought of as a fingerprint. These mass spectra, when compared against calculated spectra from a known reference sequence, can provide useful sequence information about the target. These methods accomplish fragmentation by using chemical (von Wintzingerode et al. 2002) or enzymatic means (Hartmer et al. 2003), and are specific to a mononucleotide (e.g. cleavage after every dA residue).
Mononucleotide-specific fragmentation methods are inefficient, and typically destroy much of the sequence information in the target DNA in the process of generating oligonucleotides that are short enough for analysis by mass spectrometry (Zabeau et al. 2000). This is because about 40%-50% of the target DNA is reduced to fragments four nucleotides or shorter in a typical cleavage reaction, which are too small to be informative using a MALDI-TOF instrument.
Cleavage techniques that are specific to dinucleotide sequences have been developed to overcome the limitations of mononucleotide specific fragmentation (Stanton, Jr. et al. 2003). A specific dinucleotide cleavage reaction would be expected to produce fragments with an average length of sixteen bases, which is ideal for analysis by MALDI-TOF MS. These methods utilize chemically modified nucleotide analogs (Wolfe et al. 2003) or template-directed incorporation of dinucleotide triphosphates by special polymerases (Kless 2001).
However, all these cleavage methods share a fundamental limitation: there is no way to determine the order of the bases in a fragment given only its length or molecular mass. This effectively means that existing fragmentation methods are limited to applications where a reference sequence is available so that possible fragment masses can be calculated beforehand (Bocker 2003).
It would be useful to develop methods for determining nucleic acid sequences that would require no prior sequence knowledge.